Since the enzyme electrode was first conceived, a significant interest has developed in the field of biosensors because of the simplicity and selectivity of these sensors. This increase in interest began in the 1980's, as evidenced by the publication of a new international journal Biosensors.sup.1. The rapid growth of biotechnology in the last decade has now created a demand for more and better on-line sensors that can be interfaced with computers to control and optimize bioprocesses. Numerous enzyme electrode configurations have been published, although the amount of research activity today is too great to be covered in a single review.sup.2. Present-day applications of biosensors can be found in industrial bioprocess monitoring, environmental monitoring, the food and drink industry, and clinical and in vivo application in medicine.sup.3 4. FNT .sup.1 Stoecker, P. W.; Yacynyeh, A. M. Chemically Modified Electrodes as Biosensors. Sel. Elec. Rev. 1990, 12, 137-160. FNT .sup.2 Preitag, R. Applied Biosensors. Curr. Opin. Biotech.; Anal. Biotech. 1993, 4, 75-79. FNT .sup.3 Schultz, J. S. Sophisticated Descendants of The Canary in The Coal Mine Are Based on Molecular Components of Plants and Animals Bound to Microscope Electrodes or Optical Fibers. Scientific American 1991, 264, 64-69. FNT .sup.4 Reach, G.; Wilson, G. S. Can Continuous Glucose Monitoring be Used For the Treatment of Diabetes! Anal. Chem. 1992, 64, 381-386.
The increasing commercial importance of bioreactors has stimulated research in the area of on-line monitoring of microbial and animal cell bioreactors in order to optimize performance conditions. The specificity and selectivity provided by the biological component of biosensors offer enormous potential, in principle, for continuous, on-line analysis in complex media. Despite the number of biosensor research papers published each year in the scientific literature, relatively few bioprocess sensors are commercially available. Unfortunately, many practical problems remain which have prevented the widespread application of on-line biosensors under real conditions and these have not been successfully addressed by researchers to data. In particular, the development and commercialization of biosensors has been slowed by problems of instability such as drift of the sensor signals, narrow measuring ranges for the analyte and long response time.
Short term instabilities typically result from changes in the enzyme component, such as inhibition or deactivation by components of the analyte medium. Long term instabilities (drive of the sensors signal over time) may be due to time-dependent changes in the sensor calibration constants which are caused by membrane fouling or electrode poisoning. Perm-selective membranes for amperometric biosensing have been investigated as a means to address these problems. A literature review of these membranes is provided in Wang, 1992.sup.5. FNT .sup.5 Wan, J. Permselective Coatings For Amperometric Biosensing. In Biosensors and Chemical Sensors: Optimizing Performance Through Polymeric Materials; Eddman, P. G., Wang, J., Eds.; ACS: New Mexico State University, 1992; Symposium Series 487, pp 125-132.
Although the lifetime of an enzyme in a reaction system may be prolonged in some cases, the denaturization of enzymes may be irreversible and replacement of the enzyme when the activity has degraded to an unsatisfactory degree may eventually be necessary with virtually all of the commercially available biosensors (whether on-line or not). The capability to replace only the enzyme component of the sensor without disrupting the process would not only extend the sensor operating lifetime but would allow for a substitution of other enzymes in order to change the analyte specificity of the sensor.
A few sensor systems have been described in the literature with the capacity for enzyme replacement. Brooks et al (1987/88).sup.6 and Bradley and Schmid (1991).sup.7 have described the immobilization of enzyme on graphite discs which could be replaced manually. There is no contemplation of in situ, automatic exchange of the enzyme, that is, without dismantling the sensor or interrupting the fermentation. Pieters and Bardeletti (1992).sup.8 described the immobilization of enzymes to magnetic beads which can then be manipulated using magnetic fields. This technique has been used in waste-water treatment, affinity separation processes, cell sorting, immunoassays and drug delivery. Specifically, glucose oxidase has been reversibly immobilized in an enzyme reactor coupled to a flow injection analysis system using a chain of biospecific reactions based on the binding of biotin-labelled (ie. biotinylated) antibodies to an avidin coated matrix (de Alwis and Wilson 1989.sup.9). FNT .sup.6 Brooks, S. L.; Ashby, R. E.; Turner, A. P. F.; Calder, M. R.; Clarke, D. J. Development of an On-Line Glucose Sensor For Fermentation Monitoring, Biosensors 1987/88, 3, 45-56. FNT .sup.7 Bradley, J.; Schmid, R. D. Optimization of a Biosensor For In situ Fermentation Monitoring of Glucose Concentration. Biosensor & Bioelectronics 1991, 6, 669-674. FNT .sup.8 Pieters, B. R.; Bardeletti, G. Enzyme Immobilization on a Low-Cost Magnetic Support: Kinetic Studies on Immobilized And Coimmobilized Glucose Oxidase And Glucoamylase. Enzyme Microb. Technol. 1992, 14, 361-370. FNT .sup.9 de Alwiss, U; Wilson, G. S. Strategies For The Reversible Immobilization of Enzymes by Use of Biotin-Bound Anti-Enzyme Antibodies, Talanta 1989, 36, 249-253.
In summary, at the present time, only a few reaction parameters can be reliably monitored on-line (eg. temperature, pH, dissolved oxygen tension, stir rate) without the use of highly sophisticated and costly instrumentation. The analysis of fermentation substrates, products and metabolites is usually achieved by off-line methods.sup.10. However, optimal control of a bioprocess today requires that measurable parameters be determined as frequently as possible, which in turn requires frequent sampling that increases the risk of contamination. Furthermore, off-line methods are usually to slow to be used in a closed-loop control system, that it is often difficult to ensure that samples are not significantly degraded or changed during the sampling-analysis procedure. A sensor system based on an in situ probe which could provide continuous, real-time analysis would be extremely valuable, particularly for high-density, fed-batch processes. The advantages and disadvantages of automated sampling systems in contrast with in situ probes have been discussed in the literature (Ogbomo et al, 1990.sup.11 ; Bradley et al, 1991.sup.12 ; Filippini et al, 1991.sup.13). The practical concerns involved in using an in situ biosensor probe, such as in situ sterilizability, long-term stability, adequate measuring range, and membrane fouling, have thus far prevented the widespread application and commercialization of this approach. Attempts to address these problems have heretofore been largely unsuccessful (Enfors and Molin, 1978.sup.14 ; Cleland and Enfors, 1983.sup.15 ; Bradley et al., 1988.sup.16, 1991.sup.17 and Brooks et al., 1987/88.sup.18 ; Buhler and Ingold, 1976.sup.19). In each of these probe designs, the combined functions of enzyme replacement and recalibration of the sensor cannot be performed without operator intervention. Thus, an operator must be standing by during a long fermentation run to manually replace the enzyme component periodically and then recalibrate the sensor. FNT .sup.10 Supra at 6. FNT .sup.11 Ogbomo, I.; Prinzing, U.; Schmidt, H. L. Prerequisites For The Control of Microbial Processes by Flow Injection Analysis. J. Biotechnol. 1990, 14(1), 63-70. FNT .sup.12 Bradley, J.; Stockleim, W.; Schmid, R. D. Biochemistry Based Analysis Systems for Bioprocess Monitoring and Control. Process Contr. Qual. 1991, 1, 157-183. FNT .sup.13 Filippini, C.; Sonnleitner, B.; Fiechter. A., Bradley, J.; Schmid, R. On-Line Determination of Glucose in Biotechnological Processes: Comparison Between FLA And an In situ Enzyme Electrode. J. Biotechnol. 1991, 18, 153-160. FNT .sup.14 Enfors, S. O.; Nilsson, H. Design And Response Characteristics of an Enzyme Electrode For Measurement of Penicillin in Fermentation Broth. Enzyme Microbiol. Technol. 1979, 1, 260-264. FNT .sup.15 Cleland, H., Enfors, S. O. Monitoring Glucose Consumption in an Escherichia coli Cultivation With an Enzyme Electrode. Anal. Chim. Acta. 1984b, 163, 281-285. FNT .sup.16 Bradley, J.; Anderson, P. A.; Dear, A. M.; Ashby, R. E.; Turner, A. P. F. Glucose Biosensors for the Study and Control of Baker's Compressed Yeast Production. In Computer Applications in Fermentation Technology: Modelling and Control of Biotechnological Processes; Fish, N. M., Fox, R. I., Thornhill, N. F., Eds.; Elsevier Applied Science: New York, 1988; pp 47-51. FNT .sup.17 Supra at 7. FNT .sup.18 Supra at 6. FNT .sup.19 Buhler, H., Ingold, W. Measuring pH and Oxygen in Fermenters. Process Biochem. 1976, 11(3), 19-24.
The outer membrane of a biosensor is very important, as it represents the interface between the sensor and the analyte medium. The purpose of this interface membrane is to allow the diffusion of analytes and (in electrochemical reaction) electrolytes into the investigative or analysts layer while excluding potential interfering species which may be present in the analyte medium, such as cells, proteins, inhibitors or interferents.
In U.S. Pat. No. 5,165,407 to Wilson et al., an implantable glucose sensor is provided having an enzyme immobilized on an indicating electrode and a permeable polyurethane membrane applied over the sensor body to prevent fouling of the electrode and denaturization of the enzyme. This membrane, as with others currently employed, is integrated with the sensor system and can not be replaced or reused with other systems.
The objects of this invention are to obviate or mitigate the disadvantages of the current in situ biosensor probes and the disadvantages of current membrane systems for use generally with biosensor probes.